Sindbis virus, and enveloped RNA virus, is a benign member of a large group of medically important human and veterinary pathogens. Our studies are directed at understanding the molecular mechanisms involved in viral RNA replication, virus- induced modulation of host macromolecule synthesis, and virion assembly. We have constructed a cDNA clone of Sindbis virus which can be transcribed in vitro to produce infectious RNA molecules. This cloned will be used to map rigorously the lesions in mutants which have already been characterized and to create new mutants for further study. In particular, we will map the conditional mutations responsible for specific defects in RNA synthesis and virion assembly, and therefore assign these mutant phenotypes to specific viral nonstructural or structural peptides. Of the four Sindbis nonstructural proteins, nsP4 is of particular interest. This protein contains significant sequence homology with nonstructural proteins from all plant and animal (+) strand RNA viruses examined to date, and is therefore believed to play a central role in RNA replication. We will use both site-directed and saturation mutagenesis of the infectious clone to create a new catalog of silent, lethal, and conditional mutants in nsP4. Conditional mutants will be characterized to gain a better understanding of the function of nsP4 in RNA replication. We will also study methods to improve synthesis in vitro of functional RNA transcripts and to increase the efficiency with which they can be introduced into large numbers of cells. These improvements may allow the study of lethal mutations produced by in vitro mutagenesis of the infectious clone, and will also be of use to others studying genes by in vitro transcription and transfection.